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Elucidation of forskolin biosynthetic pathway in Coleus forskohlii

Final Report Summary - BIOFORS (Elucidation of forskolin biosynthetic pathway in Coleus forskohlii)

Forskolin is a labdane type diterpenoid produced in the root of the plant Coleus forskohlii (Lamiaceae or mint family). Its medicinal activity is known since ancient times and has been used extensive in folk Indian medicine and Ayurveda. It has been shown to increase intracellular cAMP levels which then assist in the relaxation of human blood and respiratory vessels, and as such it has been proven a potent drug for treating cardiovascular problems, as well as glaucoma or asthma. Today a semisynthetic forskolin derivative has been approved for commercial use in Japan for treatment of cardiac surgery complications, heart failure and cerebral vasospasm but also 1% forskolin eye drop solution has been approved as an effective treatment for glaucoma. Although the pharmaceutical importance of forskolin, its biosynthetic route, but also the actual site of synthesis and accumulation in the plant, were not known till recently. Thus the commercially used compound is extracted from the roots of Coleus, which makes forskolin not readily available for wide use, as its purity and availability depends on plant cultivation conditions.
Consequently, in order to ensure a consistent supply of forskolin that can meet market needs, an alternative and sustainable strategy for commercial production is desirable. For addressing this issue, BioFors has been suggested a biotechnological strategy, including the production of forskolin from microbial hosts as a scalable, production platform for the cost-effective and environmentally sustainable biosynthesis of forskolin. For this purpose, necessary step would be the elucidation of forskolin biosynthetic pathway.
In that context, during BioFors, we have shown that forskolin is synthesized but also accumulates in specialized cells harboring intracellular oil body structures within the root cork (Pateraki et al., 2014). Purification and chemical analysis of coleus root oil bodies showed that forskolin as well as its hypothetical precursor, manoyl oxide (MO), are found in those structures. That was a critical step for the suggested research, as the transcriptome sequencing (RNA seq) of these specialized cells was crucial for the identification of the enzymes participating in forskolin biosynthesis.
Initially, targeted mining of the deep coleus root transcriptome resulted in the identification of a small multigene family encoding diterpene synthases (CfdiTPSs), the enzymes responsible for the formation of the chemical backbone of forskolin (MO). Expression studies of the identified genes showed that individual genes expression levels were well-correlated with the occurrence of forskolin in coleus tissues. Following functional characterization of the selected genes/enzymes, we showed that CfdiTPS2 in combination with CfdiTPS3 are involved in the biosynthesis of the correct stereoisomer of MO, forming effectively the backbone of forskolin (Pateraki et al., 2014). The next biosynthetic step towards forskolin should be the functionalization (or decoration) of MO, so from a non-active hydrocarbon will be converted to a bioactive terpenoid like forskolin. According to the chemical structure of forskolin, five oxygenations and an acetylation have to take place after MO synthesis. Currently we have identified the enzymes necessary for the synthesis of deacetyl-forskolin. They belong to the CYPs (cytochrome P450s) superfamily and more specifically to the subfamily of CYP76AHs, which is specific to Lamiaceae plant species. The first committed step is the ketone formation at carbon 11, which is catalyzed by 3 different CYPs: CYP76AH15, CYP76AH8 and CYP76AH17. Although all of them function in a similar way, we have shown that CYP76AH15 exhibits higher specificity towards the ketonated-MO. Subsequently, the multifunctional CYP76AH11 catalyzes the hydroxylation of carbons 1, 6 and 7 while CYP76AH16 hydroxylates carbon 9. Currently a more thorough characterization of the above enzymes, in respect to their substrate specificity and product formation, is taking place.
Further efforts have been made for the identification of the last enzymatic step, the acetylation of deacetyl-forskolin. Today we have identified a number of enzymes (from the acyltransferase BAHD family) able to use as substrate MO and its oxygenated derivatives in order to produce forskolin as well as a number of oxygenated MO acetylated analogues (Pateraki et al., 2015).
As it has been mentioned before, the ultimate purpose of this research was the large scale biotechnological production of forskolin. For this purpose a close collaboration has been established between BioFors researchers (PLEN, UCPH) and the biotech company Evolva S/A (http://www.evolva.com/) who is specialized for the production of high value, sustainable ingredients (like resveratrol, vanillin or the terpenoid stevia) using yeast cells as production platform. Using the technology of Evolva we have managed to incorporate efficiently into yeast genome the coleus genes (codon-optimized) responsible for the synthesis of forskolin. The functional reconstitution of the forskolin pathway in yeast has resulted in the production of high titers of deacetyl-forskolin, and lower amounts of forskolin (Pateraki et al., 2015). Further optimizations (enzymes and pathway engineering) will be necessary in order to improve forskolin titers in recombinant yeast cells.
Conclusively, BioFors has paved the way for a biotechnological, sustainable production of forskolin in yeast cells. Considering the importance of forskolin biosynthesis using biotechnology means and the impact that these finding could have to the society but also for industrial applications, BioFors results have set the ground for a close collaboration between the researchers involved with a number of other research groups and entities. Today, efforts for the incorporation of forskolin biosynthetic pathway into a number of other hosts are taking place: i) in the moss Physcomitrella patens in collaboration with Henrik Toft Simonsen, Associate Prof. in PLEN, UCPH; ii) in cyanobacteria in collaboration with the group of Prof. Poul Erik Jensen, Prof in PLEN, UCPH; in Escherichia coli in collaboration with Dr. Morten H. H. Nørholm from Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark and iv) in tobacco chloroplast in order to be able to produce forskolin exclusively in a light driven manner, from the group of Prof. Poul Erik Jensen, Prof in PLEN, UCPH (Lassen et al., 2014).
Moreover 4 provisional patents have been filled regarding forskolin biosynthesis pathway discovery and forskolin production in yeast.
Literature
Pateraki I, Andersen-Ranberg J, Hamberger B, Heskes MA, Juel Martens H, Zerbe P, Spanner Bach S, Møller BL, Bohlmann J and Hamberger B. Manoyl oxide (13R), the biosynthetic precursor of forskolin, is synthesized in specialized root cork cells in Coleus forskohlii. Plant Physiology 164: 1222–1236, (2014).
Lassen LM, Nielsen AZ, Ziersen B, Gnanasekaran T, Møller BL, and Jensen PE. Redirecting Photosynthetic Electron Flow into Light-Driven Synthesis of Alternative Products Including High-Value Bioactive Natural Compounds. Acs Synthetic Biology 3: 1-12, (2014).
Nielsen MT, Andersen-Ranberg J, Christensen U, Kristensen M, Harrison SJ, Olsen CE, Hamberger B, Møller BL, and Nørholm MHH. Microbial synthesis of the forskolin precursor manoyl oxide in enantiomerically pure form. Journal of Applied and Environmental Microbiology 80:23, 7258-7265, (2014).
Pateraki I, Andersen-Ranberg J, Jensen NB, Hallström B, Wubset S, Stærk D, Olsen CE, Møller BL, and Hamberger B. Elucidation of the biosynthetic pathway of Forskolin in Coleus forskohlii and pathway reconstitution in yeast cells. Manuscript in preparation, (2015).